Mutations in RNA Polymerase II and Elongation Factor SII Severely Reduce mRNA Levels in Saccharomyces cerevisiae

Abstract

Elongation factor SII interacts with RNA polymerase II and enables it to transcribe through arrest sites in vitro. The set of genes dependent upon SII function in vivo and the effects on RNA levels of mutations in different components of the elongation machinery are poorly understood. Using yeast lacking SII and bearing a conditional allele of RPB2, the gene encoding the second largest subunit of RNA polymerase II, we describe a genetic interaction between SII and RPB2. An SII gene disruption or the rpb2-10 mutation, which yields an arrest-prone enzyme in vitro, confers sensitivity to 6-azauracil (6AU), a drug that depresses cellular nucleoside triphosphates. Cells with both mutations had reduced levels of total poly(A) ؉ RNA and specific mRNAs and displayed a synergistic level of drug hypersensitivity. In cells in which the SII gene was inactivated, rpb2-10 became dominant, as if template-associated mutant RNA polymerase II hindered the ability of wild-type polymerase to transcribe. Interestingly, while 6AU depressed RNA levels in both wild-type and mutant cells, wild-type cells reestablished normal RNA levels, whereas double-mutant cells could not. This work shows the importance of an optimally functioning elongation machinery for in vivo RNA synthesis and identifies an initial set of candidate genes with which SII-dependent transcription can be studied.The elongation phase of transcription is an important control point for the regulation of gene expression (reviewed in references 4, 29, and 41). Several general elongation factors, including TFIIF, ELL, and elongin (SIII), are able to increase the overall rate of transcription elongation of RNA polymerase II (PolII) in vitro (7,13,26,37). SII enables PolII to transcribe through a variety of transcriptional blockages, including intrinsic arrest sites and nucleoprotein complexes (reviewed in references 29 and 41). SII reactivates arrested PolII complexes by binding to the enzyme and activating a nascent RNA cleavage activity, which eventually results in polymerase escape (reviewed in reference 28). It has been hypothesized that elongation factors such as TFIIF reduce the frequency of arrest by reducing the dwell time of PolII at arrest sites (6,15).Little is known about gene sequences that block transcription and the interaction of general elongation factors with PolII complexes in vivo. In yeast, mutation or disruption of the gene encoding SII, DST1 (gene names in this study are those designated in the Saccharomyces Genome Database; DST1 is also known as PPR2), renders cells sensitive to the base analog 6-azauracil (6AU) (19,23,24). 6AU depletes cellular levels of the RNA precursors UTP and GTP (12). Supplementing the drug-containing medium with uracil or guanine reverses this phenotype, suggesting that the drug inhibits growth because of the reduction in nucleoside triphosphate levels and thereby impairs transcription elongation (3, 12). The sensitivity of yeast to 6AU after disruption of DST1 may indicate an increased requirement by RNA polymerase for elongat...

Genetic Interactions Between TFIIF and TFIIS

“…It was previously shown that cells lacking Taf14p have a mild decrease in constitutive and induced transcription levels in vivo (Henry et al 1994), while cells disrupted in PPR2 do not exhibit overall decreased transcription levels (Lennon et al 1998). In combination with a PPR2 disruption, cells with disruptions in Figure 2.-A taf14D allele is synthetically lethal in combination with a ppr2D allele.…”

Use of an in Vivo Reporter Assay to Test for Transcriptional and Translational Fidelity in Yeast

“…2B. Here, however, a different laboratory strain of yeast was used in which the DST1 gene was disrupted by a hisG cassette (10). The paromomycin experiment shows that the assay was able to detect the induction of full-length active luciferase from transcripts containing a stop codon at position 445.…”

“…Cells lacking SII are defective in transcriptional induction of a number of genes (10,12,35). To ensure that the luciferase activity determinations were not biased by a difference in luciferase mRNA levels between the DST1 deletant and cells wild-type for DST1, we measured the amount of transcript by Northern blotting (data not shown).…”

“…can be estimated by dividing the maximal number of active luciferase molecules derived from pLuc-Stop, 1.1 ϫ 10 7 , by the number of total luciferase enzymes present in the pGAL-LUC-AAG extract, 4 ϫ 10 10 . The actual frequency would be lower if activity from the pLuc-Stop plasmid was due to other causes, one of which would be stop codon read-through.…”

Perturbation of transcription elongation influences the fidelity of internal exon inclusion in Saccharomyces cerevisiae

“…If this reflects increased competition by the downstream 3Ј splice site due to additional time for elongating polymerase to complete the downstream intron, then slowing of RNA polymerase should have the opposite effect and should suppress exon skipping (enhance exon inclusion). To test this, we used two mutations in the gene encoding the second largest subunit of RNAP II, RPB2, because multiple lines of evidence implicate this subunit in the process of transcript elongation (Treich et al 1992;Powell and Reines 1996;Lennon et al 1998). One mutant, rpb2-10, has been shown to exhibit a reduced rate of transcript elongation in vitro (Powell and Reines 1996).…”